Genomic and Physiological Traits of the Marine Bacterium Alcaligenes aquatilis QD168 Isolated From Quintero Bay, Central Chile, Reveal a Robust Adaptive Response to Environmental Stressors

Abstract

Alcaligenes aquatilis QD168 is a marine, aromatic hydrocarbon-degrading bacterium, isolated from an oil-polluted sediment of Quintero Bay, an industrial-coastal zone that has been chronically impacted by diverse pollutants. The aims of this study were to characterize the phylogenomic positions of Alcaligenes spp. and to characterize the genetic determinants and the physiological response of A. aquatilis QD168 to model environmental stressors (benzene, oxidizing agents, and salt). Phylogenomic analyses, using 35 housekeeping genes, clustered A. aquatilis QD168 with four other strains of Alcaligenes spp. (A. aquatilis BU33N, A. faecalis JQ135, A. faecalis UBA3227, and A. faecalis UBA7629). Genomic sequence analyses of A. aquatilis QD168 with 25 Alcaligenes spp., using ANIb, indicated that A. aquatilis BU33N is the closest related strain, with 96.8% ANIb similarity. Strain QD168 harbors 95 genes encoding proteins of seven central catabolic pathways, as well as sixteen peripheral catabolic pathways/reactions for aromatic compounds. A. aquatilis QD168 was able to grow on 3-hydroxybenzoate, 4-hydroxybenzoate, benzoate, benzene, 3-hydroxycinnamate, cinnamate, anthranilate, benzamide, 4-aminobenzoate, nicotinate, toluene, biphenyl and tryptophan, as sole carbon or nitrogen source. Benzene degradation was further analyzed by growth, metabolite identification and gene expression analyses. Benzene strongly induced the expression of the genes encoding phenol hydroxylase (dmpP) and catechol 1,2-dioxygenase (catA). Additionally, 30 genes encoding transcriptional regulators, scavenging enzymes, oxidative damage repair systems and isozymes involved in oxidative stress response were identified. Oxidative stress response of strain QD168 to hydrogen peroxide and paraquat was characterized, demonstrating that A. aquatilis QD168 is notably more resistant to paraquat than to H₂O₂. Genetic determinants (47 genes) for osmoprotective responses were identified, correlating with observed high halotolerance by strain QD168. The physiological adaptation of A. aquatilis QD168 to environmental stressors such as pollutants, oxidative stress and salinity may be exploited for bioremediation of oil-polluted saline sites.

Keywords: Alcaligenes; Alcaligenes aquatilis; Quintero Bay; aromatic catabolism; benzene; osmotolerance; oxidative stress; phylogenomics.

FIGURE 1

Phylogenetic analysis of representative Alcaligenes spp. strains including A. aquatilis QD168. (A) MLSA-based clustering of 26 strains of Alcaligenes spp. based on 35 housekeeping genes. Mid-rooted phylogeny showed five distinctive clades. (B) ANIb analysis of 26 strains of Alcaligenes spp. The first column indicates 95% ANI clustering (75% alignment). Isolation source is indicated in the last column. A. aquatilis QD168 belongs to cluster III.

FIGURE 2

Schematic representation of the genome of A. aquatilis QD168. Alcaligenes aquatilis QD168 genome has a size of 4,323,879 bp and contains 3,892 coding sequences, 9 rRNA and 58 tRNA. Rings from inside to outside: (1) G + C skew; (2) G + C content; (3) QD168 genes colored by functional COG class on the reverse strand; (4) predicted QD168 genes on the reverse strand; (5) predicted QD168 genes on the forward strand; (6) QD168 genes colored by functional COG class on the forward strand. Gene functions were annotated based on COG categories. The whole genome map was generated using CGView.

FIGURE 3

Cluster of orthologous groups comparison of A. aquatilis QD168 and representative Alcaligenes strains. Cluster of Orthologs (COG) classification of eight Alcaligenes spp. strains. For each COG entry the average percentage of hits among 26 Alcaligenes spp. strains has been indicated by values and dashed lines. From outer to inner circle: A. aquatilis QD168, A. aquatilis BU33N A. faecalis ZD02, A. faecalis DSM 30030^T A. faecalis subsp. phenolicus DSM 16503^T A. faecalis JQ135, A. faecalis subsp. phenolicus MB207, A. faecalis GZAF5, Alcaligenes sp. EGD-AK7.

FIGURE 4

Schematic representation of all central aromatic catabolic pathways and peripheral aromatic catabolic pathways/reactions present in A. aquatilis QD168. The inner circle (blue) includes ring-cleavage product structures of central aromatic catabolic pathways. The outer circle (green) includes the structure of aryl-CoA and dihydroxylated ring-cleavage intermediates. Dashed lines indicate multiple steps.

FIGURE 5

Functional analyses of benzene degradation by A. aquatilis QD168. (A) Predicted ben, dmp and cat genes encoding the catechol branch of the β-ketoadipate catabolic pathway and peripheral reactions on A. aquatilis QD168 genome. Genes and intergenic regions sizes are on scale (B) A. aquatilis QD168 growth on benzene (5 mM) as sole carbon and energy sources. Turbidity values were calculated as the mean ± SD of results of, at least, three independent experiments. (C) Expression of dmpP, catA and catE genes during QD168 growth on benzene (5 mM). 16S rRNA and ftsZ genes were used as reference genes. At least three independent RNA samples were collected at each condition and two independent RT-PCR reactions for each sample were done to assess reproducibility. (D) Proposed benzene degradation pathway of A. aquatilis QD168. Enzymes and compounds with experimental data are shown in green.

FIGURE 6

Genetic determinants of A. aquatilis QD168 for oxidative stress response and phylogenetic relationships of OxyR and SoxR transcriptional regulators. (A) Genetic determinants involved in oxidative stress response identified by genome mining. (B) Phylogenetic tree of OxyR protein orthologous: mid-rooted phylogeny showed the clade of QD168 OxyR (C) Phylogenetic tree of SoxR orthologous proteins: mid-rooted phylogeny showed five distinctive clades.

FIGURE 7

Oxidative stress response of A. aquatilis QD168 to H₂O₂ and paraquat. (A) Growth inhibition diameter of A. aquatilis QD168 after 60 min exposure to H₂O₂ (1–20 mM). (B) Survival of A. aquatilis QD168 after 60 min exposure to H₂O₂ (0.1–4 mM). (C) Growth inhibition diameter of A. aquatilis QD168 after 60 min exposure to PQ (1–100 mM). (D) Survival of A. aquatilis QD168 after 60 min exposure to PQ (25–500 mM). Values for growth inhibition diameter and survival of strain QD168 were calculated as the mean ± SD of results of, at least, three independent experiments. Different letters indicate significant differences between conditions (HSD Tukey test, α = 0.05) (E) Expression of oxidative stress genes of A. aquatilis QD168 cells after 60 min exposure to H₂O₂ (500 μM) and PQ (50 mM). 16S rRNA and ftsZ were used as reference genes. At least three independent RNA samples were collected at each condition and two independent RT-PCR reactions for each sample were done to assess reproducibility.

FIGURE 8

Osmotolerance response of A. aquatilis QD168 to salt stress. (A) Genetic determinants involved in biosynthesis and transport of osmo-protectant metabolites. (B) Salt tolerance of A. aquatilis QD168 in R2A medium supplemented with 0 to 10% (w v^-1) NaCl.